Biomacromolecules 2004, 5, 1926-1932
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Synthesis of Novel Biodegradable Cationic Polymer: N,N-Diethylethylenediamine Polyurethane as a Gene Carrier Tsung-fu Yang,† Wei-kuo Chin,*,† Jong-yuh Cherng,‡ and Min-da Shau‡ Department of Chemical Engineering, National Tsing-Hua University, Hsinchu 300, Taiwan, Republic of China, and Department of Applied Chemistry, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan, Republic of China Received April 21, 2004
A new cationic polymer, N,N-diethylethylenediamine-polyurethane (DEDA-PU), bearing tertiary amines in the backbone and side chains, was synthesized and used as a nonviral vector for gene delivery. The DEDAPU readily self-assembled with the plasmid DNA (pCMV-βgal) in water and buffer at physiological pH, as determined by agarose gel retardation, dynamic light scattering, zeta potential, atomic force microscopy (AFM), and restriction endonuclease protection assays. The results revealed that DEDA-PU was able to bind with plasmid DNA, yielding positively charged complexes with a size around 100 nm at a DEDAPU/DNA ratio of 50/1 (w/w). The DEDA-PU/DNA complexes were able to transfect HEK 293 cells in vitro with an efficiency comparable to a well-known gene carrier [poly(2-dimethylaminoethyl methacrylate), PDMAEMA]. The cytotoxicity of DEDA-PU was substantially lower than PDMAEMA. The degradation studies indicated that DEDA-PU degrades hydrolytically in 20 mM HEPES buffer at pH 7.4 with a half-life of approximately 60 h. This study shows that DEDA-PU holds promise as biodegradable polycations for gene delivery and is interesting candidate for further study. 1. Introduction Nonviral gene delivery systems based on complexes of condensed DNA with polycations have attracted great attention in recent years.1 The polycations/DNA complexes not only can protect DNA from nuclease degradation but also have a nanoscale size small enough to enter the cell through endocytosis. Amine-containing polycations such as poly-L-lysine (PLL),2 poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA),3 and polyethyleneimine (PEI)4 have been proposed as carriers for genetic material because they readily form complexes with DNA. However, these polycations are nonbiodegradable materials associated with a considerable degree of cytotoxicity.5,6 These deficiencies prohibit their use in some gene delivery systems where a toxicity-free carrier is required. Consequently, there have been great efforts to synthesize biodegradable polycations that can be used as gene carriers. A number of reported biodegradable gene carriers include poly(4-hydroxy-L-proline ester),7,8 poly(R-(4-aminobutyl)-L-glycolic acid),9 linear poly(β-amino ester),10,11 and polyphosphoester.12 Generally, the biodegradable polycations should be designed to incorporate hydrolytic moieties so that the polycations can readily degrade into nontoxic byproducts. As a class of biodegradable polymers, polyurethanes, because of their biodegradability and functionality in terms of chemical and physical properties, have been investigated for different biomedical applications.13-15 However, most works on poly* To whom correspondence should be addressed. E-mail: wkc@ che.nthu.edu.tw. † National Tsing-Hua University. ‡ Chia-Nan University of Pharmacy and Science.
urethanes have dealt with tissue engineering and hydrogels16 and none have focused on its use in gene delivery. Polyurethanes also have attractive features, which can be developed for the design of gene delivery carriers. These include the controlled degradation of the polymer and a great diversity of side groups and backbones (such as cationic, hydrophobic, hydrophilic), which can be introduced. In particular, adjustment of such polymer/DNA complexes hydrophilicity can be accomplished more easily for synthetic polycationic gene carriers than lipid/DNA17 systems by using copolymerization of a desirable amount of hydrophilic monomers such as poly(ethylene glycol) (PEG). The purpose of this study is to investigate whether polyurethanes are potentially suitable for gene delivery. The new water-soluble polyurethane (DEDA-PU) with tertiary amine in its backbone and side chains was successfully synthesized. To explore its qualification as a carrier, its ability on DNA condensation, endonuclease enzyme protection, cytotoxicity and transfection activity were investigated in order to assess possibilities in gene delivery. 2. Materials and Methods 2.1. Materials. L-Lysine methyl ester diisocyanate (LDI, Kyowa Hakko Kogyo, Japan) and poly(ethylene glycol) (PEG, Mw ) 200, Showa, Japan) were vacuum-dried before use. N-Methyldiethanolamine (MDEA), N,N-diethylethylenediamine (DEDA), glutaraldehyde, and 5-bromo-4-chloro3-indoyl-β-galactopyranoside (X-Gal) were purchased from Fluka Co. (Switzerland). Ethidium bromide, N-[2-hydroxyethyl] piperazine-N′-[2-ethaneslfonic acid] (HEPES), Nmethyl dibenzopyrazine methyl sulfate (electron-coupling
10.1021/bm049763v CCC: $27.50 © 2004 American Chemical Society Published on Web 07/15/2004
DEDA-Polyurethane as a Gene Carrier Scheme 1. Synthesis Scheme of DEDA-PU
reagent), and sodium(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) were purchased from Roche Co. (USA). Kpn I was purchased from Invitrogen Co. (USA). These reagents were used as received. Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA, Mw ) 3.6 × 105 g/mol) was provided by Dr. J. Y. Cherng (ChiaNan University of Pharmacy and Science). 2.2. Synthesis of Polyurethane (PU) (5). The PU was synthesized via a standard two-step process as shown in Scheme 1.18 The LDI (1) and PEG (2) with a NCO/OH molar ratio of 2/1 were mixed in anhydrous DMF solvent in a threenecked reaction flask under dry nitrogen purge and then heated to 80 °C to react. The NCO content in the mixture was periodically measured by di-n-butylamine titration18 until reduced to half the original. The chain extension was carried out by slowly adding MDEA (4) to the isocyanate-terminated prepolymer (3) until a 1/1 of NCO/OH molar ratio, using 0.5 wt % dibutyltin dilaurate as a catalyst. The mixture reacted at 80 °C until no unreacted isocyanate groups were detected. The synthesized PU (5) was precipitated by anhydrous ethyl ether and vacuum-dried at 40 °C. 2.3. Synthesis of N,N-Diethylethylenediamine-Polyurethane (DEDA-PU) (7). DEDA-PU was synthesized by using the aminolysis reaction of the PU and DEDA (6). 1.0 g PU was dissolved in 10 mL anhydrous DMF, and then an excess amount of DEDA (6) was slowly added and allowed to react at 45 °C at least 48 h. The product was precipitated in anhydrous ethyl ether and vacuum-dried at 40 °C. 2.4. Structural Characterizations. The structure of the polymers was characterized by nuclear magnetic resonance (NMR, Varian UnityInova 500-MHz spectrometer) and Fourier transform infrared (FT-IR, Perkin-Elmer 824 spec-
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troscopy). The molecular weight and distribution of polymer was determined by gel permeation chromatography analysis (GPC, Waters model LC-2410) based on polystyrene standards in THF. 2.5. Hydrolytic Degradation of DEDA-PU. DEDA-PU was dissolved in 20 mM HEPES buffer solution (pH7.4) at a concentration of 10 mg/mL and then incubated in a water bath at 37 °C. At various times during hydrolysis, the solutions of samples were dried in a vacuum for several hours to remove the water. The molecular weight of the remaining DEDA-PU was determined by high performance liquid chromatography/gel permeation chromatography (HPLC/ GPC). 2.6. Amplification and Purification of Plasmid DNA. The plasmid DNA used was pCMV-LacZ (pCMV-βgal), which contained a CMV promoter to drive the β-galactosidase (LacZ) gene expression. The plasmid DNA was amplified in Escherichia coli (DH5R strain) and purified using column chromatography (Qiagen Plasmid Mega kit, Germany). 2.7. Cell Lines and Cell Culture. Human embryonic kidney 293 cells (HEK293) were obtained from American Type Culture Collection (ATCC). The cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL Co., Ltd.) supplemented with 10% heat-inactived horse serum, streptomycin at 100 µg/mL, penicillin at 100 U/mL, 1.5 g/L sodium hydrogen carbonate, 1 mM sodium pyruvate, and 2 mM L-glutamie and maintained at 37 °C in a humidified 5% CO2-containing atmosphere. 2.8. Preparation and Characterizations of DEDA-PU/ DNA Complexes. 2.8.1. Formation of DEDA-PU/DNA Complexes. 5.0 mg/mL of the DEDA-PU was dissolved in 20 mM HEPES buffer (pH 7.4), and its serial dilutions were made. The DEDA-PU serial dilutions were added rapidly into the DNA solutions to obtain DEDA-PU/DNA complexes, in which the mass ratio of DEDA-PU/DNA (w/w) was 1/2 to 100/1. Then the complexes were allowed to selfassemble in the HEPES buffer and incubated at room temperature for 30 min before measurements. 2.8.2. DNA Gel Retardation and Restriction Endonuclease Protection Assay. The DEDA-PU/DNA complexes were loaded into a 0.7% agarose gel containing ethidium bromide (0.6 µg/mL) in a Tris-Acetate-EDTA (TAE) buffer and performed at 100 V for 90 min. After electrophoresis, the band was visualized by UV irradiation. The DEDA-PU/DNA complexes at a mass ratio of 50/1 were incubated with Kpn I at a concentration of 10 U/µL at 37 °C for 90 min in their provided reaction buffer. After the certain restriction endonuclease digesting, samples were analyzed by agarose gel electrophoresis in the same manner as described above. 2.8.3. Dynamic Light Scattering and Zeta Potential. The hydrodynamic sizes of the DEDA-PU/DNA complexes were determined by dynamic light scattering (Nicomp 380 system, U.S.A.) at 25 °C using a 5-mW He-Ne laser (λ ) 633 nm) as the incident beam at a scattering angle of 90°. The surface charges of the DEDA-PU/DNA complexes were conducted by determining the electrophoretic mobility at 25 °C with a Zeta potential system (Nicomp Instrument, USA).
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2.8.4. Atomic Force Microscopy (AFM) Images of DEDAPU/DNA Complexes. The DEDA-PU/DNA solution (2 µL) was placed on freshly cleaved ruby mica (EMS, Washington, PA).19 After 3 min of contact, the surface of the ruby mica was rinsed with 1-2 mL of distilled water and then dried with compressed air. The images of the complexes coated on the ruby mica surface were observed on a nanoscope II (Digital Instruments, Santa Barbara, CA) equipped with D scanner, operating in tamping mode and using a silicon nitride cantilever probe at room temperature. 2.9. Cytotoxicity Assay. Cytotoxicity of the DEDA-PU in comparison with the other gene carrier PDMAEMA was evaluated using the XTT assay.20 The 293 cells were cultured in complete DMEM then seeded at a density of 1 × 104 cells/well in a 96-well plate. Subsequently, the cells were incubated for 1 h at 37 °C with 200 µL of DMEM containing DEDA-PU or PDMAEMA at various concentrations. The cells were incubated in DMEM only for a negative control. After 1 h, the medium in each well was replaced with 100 µL of complete DMEM for a further 48 h incubation. Then, 50 µL of XTT labeling mixture was added to each well and incubated at 37 °C for 1 h. Cell viability was calculated from measured cell numbers by establishing the calibration curve of plating known numbers of cells (0 to 1 × 105 cells/well) 2 h prior to adding XTT reagent into wells. Results were expressed as the relatively cell viability (%) with respect to control wells containing the culture medium. 2.10. In Vitro Transfection Protocol and β-Galactosidase Assay. The 293 cells were used to evaluate the transfection efficiency of the polymer/DNA complexes. The cells were seeded in a 96-well plate (1.0 × 104 cells per well) in complete DMEM and incubated for 24 h before transfection test. Then, the polymer/DNA complexes (volume 200 µL) were added to the cells and incubated for 1 h at 37 °C. The DNA concentration was kept constant at 5 µg/mL (1.0 µg/well), and the amounts of polymer/DNA were expressed as weight/weight ratios. The medium was then replaced with complete DMEM and the cells were incubated for 48 h. Expression of the pCMV-βgal gene was established by incubation of fixed cells (0.25% glutaraldehyde; 5 min at 4 °C) with 0.8 mg/mL X-Gal solution for 24 h at 37 °C.21 Using a light microscope, transfected cells were made visible as blue spots and were quantified by counting the number of blue spots in each well. Besides evaluation of the number of transfected cells, the relative number of living cells was determined using the XTT assay. 3. Results and Discussion 3.1. Structural Characterizations of PU (5) and DEDAPU (7). Figure 1 showed the FT-IR spectra of the synthesized PU and DEDA-PU. The peaks at 1720 cm-1(CdO stretching, urethane), 1662 cm-1 (CdO stretching, amide), 1535 cm-1 (N-H bending, amide), and 3340 cm-1(N-H stretching, urethane) represent the absorptions of urethane links in the PU and DEDA-PU. The pendent group of the DEDA-PU contained an amide linkage. This caused stronger band absorptions of the amide group, such as CdO stretching
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Figure 1. FT-IR spectra of (a) PU and (b) DEDA-PU.
(1662 cm-1, amide) and N-H bending (1535 cm-1, amide), in the DEDA-PU than in the PU. The chemical shifts of characterized protons are listed in Tables 1 and 2. These shifts are based on the assigned labels of proton in the chemical structures of the PU and DEDAPU as indicated. Tables 3 and 4 list the chemical shifts of characterized carbon in PU and DEDA-PU. These shifts were based on the assigned labels of carbon in the chemical structures of the PU and DEDA-PU as indicated. The difference in the 1H NMR spectra between PU and DEDAPU was that there were two chemical shifts of 0.9 and 7.0 ppm that appeared in the DEDA-PU, derived from the methyl and amide protons of the pendant group of DEDA-PU, respectively. An identical observation was made in the 13C NMR spectra, showing two chemical shifts at 10.8 and 162.8 ppm, representing the methyl and carbonyl carbons of the pendant group in the DEDA-PU, respectively. There was also accompanied with a disappearance of chemical shift at 172.2 ppm, which represented the ester carbon of the pendant group in the PU. The FT-IR, 1H NMR, and 13C NMR characterizations of the synthesized polymers (5 and 7) provided clear evidence that the DEDA-PU had been successfully synthesized through the aminolysis reaction of the PU and DEDA. In addition, the GPC data of the DEDA-PU showed that the weight-averaged molecular weight of the DEDA-PU was 12 600 with a polydispersity of 1.56, relative to polystyrene standards in THF. 3.2. In Vitro Hydrolytic Degradation of DEDA-PU. The hydrolytic degradation behavior of the DEDA-PU in the 20 mM HEPES buffer was conducted in the condition of pH 7.4 at 37 °C. Figure 2 showed the reduction of molecular weight of the DEDA-PU while dissolved in the 20 mM HEPES buffer at various times. There is a continuous decrease of molecular weight (Mw) of the DEDA-PU. The rate of degradation of the DEDA-PU during the first 24 h was comparatively slower than the subsequent 48 h. Since the degraded polymer had shorter chains and more hydroxyl and amine end groups, they could accelerate degradation rates via nucleophilic attacks on the urethanes.22 Moreover, an introduction of PEG segments into the backbone structure increased the hydrophilicity and flexibility of the polymer,
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DEDA-Polyurethane as a Gene Carrier Table 1. 1H NMR Data of PU
chemical structure of PU type of proton chemical shifts (ppm)
1 3.6
2 4.1∼4.2
3 2.6∼2.9
4 2.3
5 3.1
6,7,8 1.3∼1.8
9 3.6
a 5.7
b 5.3
Table 2. 1H NMR Data of DEDA-PU
chemical structure of DEDA-PU type of proton chemical shifts (ppm) Table 3.
13C
1 3.6
2 4.1∼4.2
3 2.6∼2.9
4 2.3
5 3.1
6,7,8 1.3∼1.8
9 3.6
10 0.9
a 5.7
b 5.3
c 7.0
NMR Data of PU
chemical structure of PU aliphatic carbons 1 63.8∼70.4 Table 4.
13C
2 58.8
3 56.1
4 43.7
5 53.6
6 31.8
7 29.2
8 22.3
9 40.4
10 62.1
carbonyl carbons a b 156.6 156.1
ester 172.2
NMR Data of DEDA-PU
chemical structure of DEDA-PU aliphatic carbons 1 63.8∼70.4
2 58.8
3 56.1
4 43.7
5 53.6
promoting the susceptibility to hydrolysis.23 Therefore, a shorter DEDA-PU containing PEG segments speeded the degradation rate of the polymer. 3.3. Size Analysis and Morphology of the DEDA-PU/ DNA Complex. The DEDA-PU and DNA (pCMV-βgal) were mixed in the 20 mM HEPES buffer to form the selfassembling complexes. To be useful gene carriers, polycations must also be able to self-assemble plasmid DNA into polymer/DNA complexes small enough to enter a cell through endocytosis. For most cell types, this size requirement is on the order of 200 nm or less.2 Figure 3 showed
6 31.8
7 29.2
8 22.3
9 40.4
11 42.1
12 10.8
carbonyl carbons a b amide 156.6 156.1 162.8
the size of the DEDA-PU/DNA complexes at various mass ratios ranging from 1/2 to 100/1, determined by the dynamic light scattering (DLS). With the low DEDA-PU content, the size of complexes decreased with the increase of mass content of the DEDA-PU until the mass ratio of DEDAPU/DNA reached 20/1. With the further increase of mass content of the DEDA-PU, the size of complexes slightly increased, proportionally to the mass content of the DEDAPU. The average diameters (100-200 nm) of the complexes described above (except mass ratio of 1/2) fall within the general size requirements for cellular endocytosis. Figure 4
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Figure 2. Hydrolytic degradation of DEDA-PU incubated in 20 mM HEPES buffer at pH 7.4 and 37 °C. Degradation is expressed as percent degradation over time based on GPC data.
Figure 3. Sizes of polymer/DNA complexes formed from pCMV-βgal plasmid and DEDA-PU as a function of polymer concentration.
showed the atomic force microscopy (AFM) images of DEDA-PU/DNA complexes at mass ratios of 1/1 and 5/1. At the low mass ratio of DEDA-PU/DNA (1/1), pCMV-βgal was only partially condensed by DEDA-PU, therefore, the coiled chains of pCMV-βgal were still observed as shown in Figure 4a. By increasing the mass ratio of DEDA-PU/ DNA to 5/1, nearly all complexes condensed to roughly spherical shape as shown in Figure 4b and the discretely spherical images verified the impression of very efficient DNA condensation by the DEDA-PU. The AFM image of complexes was observed after the complexes were coated on the mica and dried with compressed air. The observed dimension, therefore, was different from the one determined in aqueous media using DLS. The former was 185 ( 15 nm and the latter was 148 ( 14 nm. 3.4. Zeta-Potential Analysis of the DEDA-PU/DNA Complex. The surface charges of the DEDA-PU/DNA complexes were conducted by determining the electrophoretic mobility at 25 °C with the zeta-potential analysis. Figure 5 showed the zeta potential changes with the mass ratios of DEDA-PU/DNA complexes ranging from 1/2 to 100/1. The free DNA possessed a negative zeta-potential of -47 mV. The zeta-potential of the resulting complex changed from a negative charge to a positive charge when the amount of DEDA-PU was increased. When the mass ratio
Figure 4. AFM images of DEDA-PU/pCMV-βgal (w/w): 1/1(a) and 5/1(b) complexes. The height of the AFM image is more than 15 nm above the ruby mica. The x and y dimensions are scaled as shown.
Figure 5. Zeta potentials of polymer/DNA complexes formed from pCMV-βgal plasmid and DEDA-PU as a function of polymer concentration.
of DEDA-PU/DNA complexes was higher than 50/1, the surface of pCMV-βgal was fully occupied with the DEDAPU molecules to form positive charge complexes. The
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Figure 7. Cytotoxicity profiles of DEDA-PU and PDMAEMA. Viability of HEK293 cells is expressed as a function of polymer concentration.
Figure 6. DNA gel retardation and restriction endonuclease protection assay of DEDA-PU. Lanes: (1) 200 ng pCMV-βgal; (2) 200 ng pCMV-βgal treated with Kpn0; (3) DEDA-PU/pCMV-βgal (w/w):1/1; (4) DEDA-PU/pCMV-βgal (w/w):20/1; (5)DEDA-PU/pCMV-βgal (w/ w):50/1; (6) DEDA-PU/pCMV-βgal (w/w):50/1 treated with Kpn0.
complexes with extra positive charges on their surfaces allowed better interaction with the target cell membrane, resulting in an enhanced uptake. 3.5. Gel Retardation and Restriction Endonuclease Protection Assay of DEDA-PU/DNA Complexes. Figure 6 illustrated the electrophoretic mobility behaviors of DEDAPU/DNA complexes on agarose gel electrophoresis. DEDAPU was dissolved in sterilized water at the concentration of 1.0 µg/µL. A fixed amount of DNA (200 ng) was incubated with DEDA-PU solutions at desired DEDA-PU/DNA mass ratios for 30 min. The protection effect of DEDA-PU to plasmid DNA in this study was demonstrated using Kpn I as a model enzyme. The treatments of free DNA and DNA with Kpn I were shown in lanes 1 and 2. Lanes 3, 4, and 5 were the DEDA-PU/DNA complexes with the mass ratios of 1/1, 20/1, and 50/1, and lane 6 was the DEDA-PU/DNA complexes (mass ratio of 50/1) in which was treated with Kpn I. DNA was partially retained by the presence of DEDAPU at a mass ratio of 1/1 (lane 3), 20/1 (lane 4) and totally retained at a mass ratio of 50/1 (lane 5). This result revealed that amine groups of DEDA-PU carrying positive charges interacted with DNA phosphate groups with negative charges to form neutral-close self-assembly complexes. The plasmid DNA was incubated with the restriction endonucleases Kpn I at a concentration of 10 U/µL, which cleaved the plasmid DNA in the CMV-promoter region or in the ampicillin resistance region. The Kpn I treated plasmid DNA generated the pure linear plasmid form as shown in lane 2. However, it was observed that the plasmid DNA was completely protected against Kpn I when the presence of DEDA-PU in complexes was at a mass ratio of 50/1 as indicated in lane 6. 3.6. Cytotoxicity of DEDA-PU. Cumulative cellular exposure time plays a primary role in the cytotoxicity of nondegradable or slowly degradable polycations.24 The
cytotoxicity of the polycations may also influence the gene expression in interfering with transcription and translation processes in the cells. To determine the cytotoxicity of the DEDA-PU in comparison with PDMAEMA, a well-known gene carrier, we performed a XTT assay using the HEK293 cell line. Cells were incubated with increasing amounts of DEDA-PU (ranging from 2.5 to 500 µg/mL) or PDMAEMA (ranging from 2.5 to 150 µg/mL). Figure 7 showed that DEDA-PU exhibited lower toxicity on 293 cells than PDMAEMA. The IC50 [defined as the concentration resulting in 50% inhibitory activity (cell death)] for the PDMAEMA was around 30 µg/mL, whereas DEDA-PU showed more than 80% cell viablity even at a higher dose (500 µg/mL) with no significant change in cell morphology and proliferation relative to controls. Nonetheless, the mechanisms of polycations-induced cell death with both biochemical and biophysical mechanisms is poorly understood.25-27 A more direct structure/function-based comparison between DEDAPU and PDMAEMA cannot be made due to differences in polymer structure and molecular weight. We infer that the DEDA-PU can provide better cytotoxicity profiles than presently used gene carrier (PDMAEMA) due to the introduction of PEG segments for the DEDA-PU backbone alternately to reduce the high charge density of the polymer. 3.7. In Vitro Transfection. The transfection efficiency of DEDA-PU/DNA complexes was performed by an indication of β-galactosidase expression. The transfection efficiency is a function of multiple parameters including polymer/DNA composition, DNA concentration, transfection time, etc. We mainly investigated the effect of DEDA-PU/DNA (w/w) ratios in HEK 293 cells, and the results were shown in Figure 8a. It can be seen that DEDA-PU promoted the cellular uptake and subsequent expression of the transgene. The number of transfected cells showed a bell-shaped curve dependence on the DEDA-PU/DNA (w/w) ratios, as observed earlier for other polymer-based transfection systems.28,29 The transfection efficiency increased with higher DEDA-PU/DNA ratios (w/w), reaching optimal transfection efficiency at a ratio of 50/1. The optimal transfection efficiency of DEDA-PU/DNA complexes occurred at where plasmid DNA was condensed with DEDA-PU into a small particle and positively charged complexes. Also, the trans-
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Acknowledgment. The authors acknowledge with gratitude financial support from the National Science Council of the Republic of China through Grant No. NSC 90-2216-E041-006. References and Notes
Figure 8. Effect of polymer concentration on the number of transfected cells (8a) and on the relative cell viability (8b). Transfection was performed in HEK293 cells for 1 h at a 1 µg DNA per well.
fection efficiency of DEDA-PU/DNA complexes was shown as nearly equivalent to that of PDMAEMA in 293 cells. Furthermore, the cytotoxicity of DEDA-PU/DNA complexes was still lower than that of PDMAEMA/DNA complexes (Figure 8b). 4. Conclusions To summarize, a new water-soluble polyurethane with tertiary amines in the backbone and side chains was synthesized. The polymer, DEDA-PU, is likely to be nontoxic and able to transfect to mammalian cells within 48 h. The presence of a PEG segment remarkably decreased the cytotoxicity of the polycations. Also, the DEDA-PU was quickly degraded, minimizing the cumulative cytotoxic effect from a cationic polymer. These biodegradable polycations with pendant charged groups, backbone charged groups, and PEG segments in the backbone hold promise as new biodegradable polycations for gene delivery and are interesting candidate for further study.
(1) Kabanov, A.; Felgner, P.; Seymour, L. Self-assembling complexes for gene deliVery from laboratory to clinical trial; John Wiley & Sons: New York, 1998. (2) Zauner, W.; Ogris, M.; Wanger, E. AdV. Drug DeliVery ReV. 1998, 30, 97-113. (3) Vande, W.; Cherng, J. Y.; Talsma, H.; Hennink, W. E. J. Controlled Release 1997, 49, 59-69. (4) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. 1995, 92, 72977301. (5) Deshmukh, H. M.; Huang, L. New J. Chem. 1997, 21, 113-124. (6) Brazeau, G. A.; Attia, S.; Poxon, S.; Hughes, J. A. Pharm. Res. 1998, 15, 680-684. (7) Lim, Y.; Kim, C.; Kim, K.; Kim, S. W.; Park, J. J. Am. Chem. Soc. 2000, 122, 6524-6525. (8) Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658-3662. (9) Lim, Y.; Han, S.; Kong, H.; Lee, Y.; Park J.; Jeong, B.; Kim S. Pharm. Res. 2000, 17, 811-816. (10) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761-10768. (11) Akinc, A.; Lynn, D. M.; Anderson, D. G.; Langer, R. J. Am. Chem. Soc. 2003, 125, 5316-5323. (12) Wang, J.; Mao, H.; Leong, K. J. Am. Chem. Soc. 2001, 123, 94809481. (13) Lamba, M.; Woodhouse, K.; Cooper, S. Polyurethane in biomedical applications; CRC Press: Boca Raton, FL, 1998. (14) Gogolewski, S. Biomedical polyurethanes- Desk reference of functional polymers; American Chemical Society: Washington, DC, 1997. (15) Zhang, J. Y.; Beckman, E. J.; Piesco, N. P.; Agarwal, S. Biomaterials 2000, 21, 1247-1258. (16) Nathan, A.; Bolikal, D.; Vyavahare, N.; Samuel; Kohn, J. Macromolecules 1992, 25, 4476-4484. (17) Sato, T.; Kawakami, T.; Shirakawa, N.; Okahata, Y. Bull. Chem. Soc. Jpn. 1995, 68, 2709-2715. (18) Hepburn, C. Polyurethane Elastomer, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 1992. (19) Hansma, H. G.; Golan, R.; Hsieh, W.; Lollo, C. P.; Ley, P. M.; Kowh, D. Nucleic Acids Res. U.S.A. 1998, 26, 2481-2487. (20) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827-4833. (21) Lim, K.; Chae, C. B. Biotechniques 1989, 7, 576-579. (22) Mehrdad, M.; Nasser, S. S. Polym. degrad. Stab. 2003, 80, 199202. (23) Skarja, G. A.; Woodhouse, K. A. J. Biomat. Sci., Polym. Ed. 1998, 9, 271-295. (24) Choksakulnimitr, S.; Masuda, S.; Tokuda, H.; Takakura, Y.; Hashida, M. J. Controlled Release 1995, 34, 233-241. (25) Antonelli, M.; Olate J.; Allende, C. C.; Allende, J. E. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1991, 99, 827-832. (26) de Kruijff B., B.; Rietveld, A.; Telders, N.; Vaandrager, B. Biochim. Biophys. Acta 1985, 820, 295-304. (27) Gad, A. D.; Silver, L.; Eytan, G. D. Biochim. Biophys. Acta 1982, 690, 124-132. (28) Cherng, J. Y.; van de Wetering, P.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. Pharm. Res. 1996, 13, 1038-1042. (29) Gebhart, C. L.; Kabanov A. V. J. Controlled Release 2001, 73, 401-416.
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